Introduction: Despite an information explosion about resistance mechanisms in bacteria, patients are dying of untreatable infections for the first time since antibiotics were discovered. The main cause of multidrug resistance (MDR) in gram-negative bacteria is active efflux of drug by bacterial pumps regulated by a complex network of transcription factors (TFs). Understanding this regulation is the key to understanding MDR development: bacteria can become MDR simply by increasing expression of these pumps. Significance: Bacteroides fragilis (BF) are ubiquitous gut commensals that are important in many aspects of human health and disease. They cause devastating disease when they escape the gut and their resistance to antimicrobials is increasing significantly. Mortality due to untreated BF bacteremia is >50%! We hypothesize that MDR in BF can be caused by efflux pumps that are regulated by (TFs). Further, we hypothesize that these TFs may be important in virulence. Preliminary Studies: Previously, we identified and characterized the BF bme efflux pump genes and showed that they were implicated in clinical MDR (described in 18 publications) and demonstrated that increased pump levels and MICs could be induced by a variety of agents and stressors. Research Plan: Our questions: 1) Which TFs are the most important in MDR? 2) What phenotype is associated with TF? Is regulation direct or indirect? 3) Do TFs that indirectly regulate pump genes directly regulate other genes? Does another TF bind to promoter region of [unreadable]orphan[unreadable] bme pump gene? 4) Are TF transcript levels increased in MDR clinical isolates? Does biofilm forming ability correlate with TF transcript levels in clinical isolates? Does exposure to bile in the gut affect TF transcription? Aim 1: Identify the activator and repressor TFs most important in pump regulation and MDR using a functional genomic approach. The TFs with the most dramatic changes (along with several TF candidates [unreadable]in hand[unreadable]) will be further characterized in AIM 2. AIM 2: Characterize TFs most important in efflux-mediated MDR using phenotypic assays, binding studies, and description of the TF regulon. AIM 3: Indirect regulation of efflux associated RND by TFs. 3A. Identify genes directly regulated by [unreadable]Indirect TFs[unreadable] identified in AIM 2. 3B. Identify TFs that directly regulate [unreadable]orphan[unreadable] pump genes identified in AIM 2. AIM 4: TFs in the natural environment: TF levels in clinical isolates and the effects of metronidazole or bile exposure on TF levels in BF. This proposal will take advantage of our unique resources: expertise with molecular, genetic and biochemical approaches, a massive collection of clinical isolates and associated MIC data, and excellent collaborative relationships with a world-class group of experts. A successful outcome of these studies will elucidate the modes of regulation of MDR efflux pumps, will lead to the development of new strategies to avoid and overcome MDR and will facilitate other molecular studies of B. fragilis.